Identification of cuproptosis-related gene SLC31A1 and upstream LncRNA-miRNA regulatory axis in breast cancer

Mounting evidence indicate that cuproptosis, a novel form of programmed cell death, contributes to cancer development and progression. However, a comprehensive analysis regarding the expressions, functions, and regulatory network of cuproptosis-related genes is still lacking. In the present work, cuproptosis-related genes, upstream miRNAs and lncRNAs, and clinical data of breast cancer from TCGA database were analyzed by R language including Cox regression analysis, correlation calculation, ROC curve construction, and survival evaluation, and were further verified by public-available databases. Chemosensitivity and immune infiltration were also evaluated by online tools. SLC31A1 was significantly increased in breast cancer samples than those in normal tissues. SLC31A1 was negatively related to a favorable outcome in breast cancer, and the AUC value increased with the prolongation of follow-up time. LINC01614 and miR-204-5p were potential upstream regulators of SLC31A1. Moreover, SLC31A1 was significantly positively correlated with different immune cells infiltration, immune cell biomarkers, and immune checkpoints in breast cancer. SLC31A1 was a potential cuproptosis-related gene in breast cancer, which was significantly upregulated and was able to predict diagnosis, prognosis, chemosensitivity, and immune infiltration. LINC01640/miR-204-5p/SLC31A1 might be a significant and promising axis during cuproptosis in breast cancer.


Survival analysis of cuproptosis-related genes in breast cancer
Prognostic values of two cuproptosis-related genes in breast cancer, namely CDKN2A and SLC31A1, were then evaluated.First, overall survival analysis was performed by using the survival package in R language.In particular, no statistical prognostic value of CDKN2A in breast cancer was observed (Fig. 2A), while patients with higher expression of SLC31A1 displayed a poor prognosis (Fig. 2B).GEPIA was utilized to carry out overall survival analysis.For CDKN2A, no statistical difference of overall survival was found between lower group and higher group in breast cancer (Fig. 2C).However, group with lower expression of SLC31A1 exhibited a better overall survival, with respect to group with higher expression of SLC31A1 (Fig. 2D).Kaplan-Meier plotter was also applied for verification.Only SLC31A1, but not CDKN2A, was negatively related to a favorable overall survival in breast cancer (Fig. 2E, F).Therefore, SLC31A1 was chosen for further investigation.Two GEO datasets, GSE12276 and GSE19615, were introduced to verify the predictive value of SLC31A1 in prognosis by using PrognoScan.The results showed that breast cancer patients with high expression of SLC31A1 had a poor relapse free survival (Fig. 3A-E) and distant metastasis free survival (Fig. 4A-F).Taken together, these findings suggest that SLC31A1 might be the most potential cuproptosis-related gene and could be a promising unfavorable prognostic biomarker in breast cancer.

Expression, PPI network, enrichment analysis, and ROC curve of SLC31A1
Gene expression of SLC31A1 in breast cancer samples and adjacent normal tissues were analyzed.Expression level of SLC31A1 was increased in breast cancer samples with respect to normal tissues (Supplementary Fig. 1A).Protein-protein interaction information and networks (Supplementary Fig. 1B) for SLC31A1 gene were analyzed by STRING database and Cytoscape software, including CCS, SLC11A2, ATP7A, ATP7B, ATOX 1, SLC22A2,   www.nature.com/scientificreports/SLC22A1, SLC30A1, CP, and COX 17. Enrichment pathway analysis showed that the genes were related to copper ion binding and transport (Supplementary Fig. 1C, D).In order to further evaluate the diagnostic value of cuproptosis-related gene SLC31A1 in breast cancer, ROC curve of SLC31A1 was drawn by using the survival package and timeROC package in R language based on breast cancer samples and normal breast tissues from TCGA.According to the ROC curve, SLC31A1 displayed the ability to distinguish breast cancer samples from normal breast tissues, and the AUC value increased with the prolongation of follow-up time (Fig. 3F).Specifically, AUC of 1 year, 5 year, and 15 year was 0.542, 0.574, and 0.666, indicating that SLC31A1 might also be a promising diagnostic marker in breast cancer.

Prediction of upstream lncRNAs of miR-204-5p/SLC31A1 in breast cancer
Potential upstream lncRNAs of the miR-204-5p/SLC31A1 axis were subsequently predicted.As described above, differentially expressed lncRNAs of breast cancer were comprehensively listed using the limma package in R language, and univariate Cox regression analysis was performed on the obtained 137 up-regulated lncRNAs.A total of 32 lncRNAs were enrolled, followed by Pearson correlation analysis between miR-204-5p and 32 lncRNAs (Table 3).Prognostic values were then analyzed on these 32 lncRNAs by the survival package in R language.The results demonstrated that breast cancer patients with increased levels of AC009686.2,AC132807.2,AC093515.1,AC129926.2,AL513123.1,RHPN1-AS1, KCNMB2-AS1, LINC01614, LINC01705, and C6orf99 had a better overall survival (Fig. 7A-J).Correlation analysis between SLC31A1 and the obtained 10 lncRNAs indicated that LINC01614 was most highly positively correlated with SLC31A1 (Table 4).Collectively, LINC01614/miR-204-5p/ SLC31A1 might be a potential axis related to cuproptosis in breast cancer.

Role of SLC31A1 in chemosensitivity and immune response
Since cuproptosis-related gene SLC31A1 was highly expressed and served as a promising diagnostic and prognostic marker in breast cancer, it is necessary to evaluate the role of SLC31A1 in predicting chemotherapeutic response during breast cancer treatment.By using the online tool GSCA, it was found that SLC31A1 modulated the chemosensitivity of multiple drugs (Fig. 8A).In terms of breast cancer, SLC31A1 expression was markedly decreased in non-response groups compared with response groups, when patients were treated with widely used chemotherapy regimens including FEC (fluorouracil, epirubicin, cyclophosphamide) plus docetaxel (Fig. 8B),   www.nature.com/scientificreports/TA (taxane, anthracycline) (Fig. 8D), and FEC (fluorouracil, epirubicin, cyclophosphamide) plus paclitaxel (Fig. 8F).Moreover, AUC was 0.854 (Fig. 8C), 0.746 (Fig. 8E), and 0.729 (Fig. 8G) in the corresponding chemotherapy regimens, revealing that SLC31A1 was able to distinguish non-response groups from response groups in breast cancer.Role of SLC31A1 in immune response was also determined.Interestingly, levels of infiltrated immune cells gradually elevated along with the increase of SLC31A1 copy number (Fig. 9A).In particular, SLC31A1 was significantly associated with infiltration degree of CD4 + T cells, macrophages, neutrophils, and dendritic cells.Besides, correlation analysis of SLC31A1 and immune cells biomarkers indicated that SLC31A1 was positively correlated with various immune cells biomarkers (Table 5).Given that immune checkpoints are closely related to immune response, the relationship between SLC31A1 and several immune checkpoints including PDCD1, CD274, and CTLA4 were therefore analyzed.Results from TIMER (Fig. 9B-D), GEPIA (Fig. 9E-G), and star-Base (Fig. 9H-J) showed that CD274 and CTLA4, but not PDCD1, were significantly positively correlated with SLC31A1 expression.Consequently, cuproptosis-related gene SLC31A1 showed a vital role in predicting chemosensitivity and immune response.

Discussion
Breast cancer is the most common cancer among women worldwide 1 .Precise surgery, adjuvant chemotherapy, and even immune therapy have greatly improved the overall survival, but prognosis of breast cancer patients remains unsatisfied 2 .Current anti-cancer therapy is based on clinical, pathological, and molecular features, and a novel approach to predict diagnosis, prognosis, and chemosensitivity is urgently required.Therefore, discovering some more promising and effective biomarkers and a significant signaling axis is of crucial importance.
Cuproptosis is a novel form of programmed cell death triggered by accumulation of intracellular copper level and aggregation of mitochondrial lipoylated proteins 3 .Mounting evidence indicate that cuproptosis contributes to cancer development and progression 4 .However, a comprehensive analysis regarding the expression patterns, diagnostic and prognostic values, and regulatory network of cuproptosis-related genes is still lacking and need to be deeply investigated.By using R language and multiple online databases on 17 candidate cuproptosis-related genes, CDKN2A and SLC31A1 were found to be increased in breast cancer samples with respect to normal tissues.After analyzing and confirming their prognostic values in breast cancer, SLC31A1 was selected as the most potential gene responsible for cuproptosis.SLC31A1 is a trans membrane protein maintaining copper homeostasis that has been recently reported to negatively contribute to cisplatin resistance in various cancers including osteosarcoma and epithelial ovarian cancer 25,26 .In the present study, SLC31A1 expression was significantly decreased in non-response groups with respect to response groups, when breast cancer patients were treated with conventional chemotherapy regimens including FEC plus docetaxel, TA, and FEC plus paclitaxel.Opposite to the above results, Takeda et al. 27 reported that SLC31A1 was predominantly expressed in triple-negative breast cancer resistant to TA-based neoadjuvant chemotherapy.Thus, there is a major need to evaluate SLC31A1 expression in different molecular subtypes of breast cancer treated with different chemotherapy regimens.
Research over the past few years has indicated that miRNAs are a class of single-stranded and noncoding RNA with a capacity for posttranscriptional regulation of gene expression 24 .In the present work, upstream miRNAs of SLC31A1 were comprehensively predicted, after which Cox regression analysis, correlation calculation, ROC curve construction, and survival evaluation were performed.Among the candidate miRNAs, miR-204-5p was considered as the most potential upstream miRNA of cuproptosis-related gene SLC31A1.Several publications have shown that miR-204-5p acts as a tumor suppressor in many types of cancer including colorectal cancer, glioma, and thyroid carcinoma [28][29][30] .However, in breast cancer, studies on miR-204-5p expression show conflicting results, suggesting a possible dual regulatory role.While some investigations supported a tumor suppressor role of miR-204-5p in regulating proliferation, metastasis, and immune microenvironment remodeling in basal-like breast cancer cells 31 , other studies showed up-regulation of miR-204-5p in breast cancer tissues and the proproliferative effect of miR-204-5p in luminal breast cancer cells 32 .Given that no relevant reports on miR-204-5p and SLC31A1 are currently available, future studies investigating the regulation of miR-204-5p in SLC31A1 are needed in different breast cancer subtypes with distinctly different biology.
In recent years, lncRNAs have been reported to take part in various biological processes via regulating multiple molecules including miRNAs 33 .In the present work, LINC01614 was identified as the potential upstream lncRNA of miR-204-5p, after a series of lncRNAs prediction, Cox regression analysis, correlation calculation, and survival evaluation.Previous studies have showed that LINC01614 functioned as an oncogenic lncRNA in several malignancies, such as pancreatic cancer, papillary thyroid carcinoma, and gastric cancer [34][35][36] .In breast cancer, lncRNA transcriptional landscape identified LINC01614 as a non-favorable prognostic biomarker.Highest expression of LINC01614 was observed in luminal and HER2 + breast cancer subtypes, while lowest expression was in basal-like breast cancer subtype 37 .Moreover, LINC01614 was found to regulate epithelial-mesenchymal transition and tamoxifen sensitivity in luminal breast cancer cells 38 .Up to now, there is no direct evidence reporting the regulation of LINC01614 on miR-204-5p.It is therefore desired that further attention be drawn to this field.
Tumor microenvironment, composed of cancer cells, surrounding fibroblasts, and tumor-infiltrating immune cells, play an essential role in oncogenesis and immunotherapy efficacy 39 .In the present study, SLC31A1 was significantly associated with infiltration degree of immune cells and levels of immune cells biomarkers.In addition,

Figure 2 .
Figure 2. Overall survival analysis of CDKN2A and SLC31A1 in breast cancer.Prognostic values of CDKN2A (A) and SLC31A1 (B) in breast cancer were analyzed by the survival package in R language.Prognostic values of CDKN2A (C) and SLC31A1 (D) in breast cancer were accessed by GEPIA.Prognostic values of CDKN2A (E) and SLC31A1 (F) in breast cancer were measured by Kaplan-Meier plotter.

Figure 3 .
Figure 3. Prognostic value of SLC31A1 in breast cancer verified using GSE12276 dataset.Expression plot (A), expression histogram (B), P-value plot (C), Kaplan-Meier plot for relapse free survival (D), survival time plot (E), and ROC curve of 1, 5, and 15 years of SLC31A1 (F) in breast cancer patients of GSE12276 dataset were analyzed by PrognoScan.

Figure 4 .
Figure 4. Prognostic value of SLC31A1 in breast cancer verified using GSE19615 dataset.Expression plot (A), expression histogram (B), P-value plot (C), Kaplan-Meier plot for distant metastasis free survival (D), survival time plot (E), and attribute plot (F) of SLC31A1 in breast cancer patients of GSE19615 dataset were analyzed by PrognoScan.

Figure 8 .
Figure 8. Role of SLC31A1 in chemosensitivity.(A) Chemosensitivity of multiple drugs regulated by SLC31A1 was predicted by GSCA.SLC31A1 expression (B) and AUC value (C) in patients treated with FEC plus docetaxel regimen was evaluated by CTR-DB.SLC31A1 expression (D) and AUC value (E) in patients treated with TA regimen was evaluated by CTR-DB.SLC31A1 expression (F) and AUC value (G) in patients treated with FEC plus paclitaxel regimen was evaluated by CTR-DB.

Figure 9 .
Figure 9. Role of SLC31A1 in immune response.(A) Levels of infiltrated immune cells under various copy number of SLC31A1 in breast cancer were analyzed by TIMER.Correlation analysis of SLC31A1 and immune checkpoints PDCD1 (B), CD274 (C), and CTLA4 (D) in breast cancer were calculated by TIMER.Correlation analysis of SLC31A1 and immune checkpoints PDCD1 (E), CD274 (F), and CTLA4 (G) in breast cancer were determined by GEPIA.Correlation analysis of SLC31A1 and immune checkpoints PDCD1 (H), CD274 (I), and CTLA4 (J) in breast cancer were analyzed by starBase.

Table 1 .
Expressions of cuproptosis-related genes in breast cancer analyzed by R. *P-value < 0.05; **P-value < 0.01; ***P-value < 0.001; values in bold indicate that these cuproptosis-related genes are upregulated in breast cancer and these results are statistically significant.

Table 2 .
Correlation between SLC31A1 and 8 predicted miRNAs in breast cancer analyzed by R.

Table 4 .
Correlation between SLC31A1 and 10 predicted lncRNAs in breast cancer evaluated by R.